I. Sepsis PA0 II. Prevention And Treatment PA0 III. Inhibiting Cytokines Released During Sepsis PA0 IV. Prophylaxis
Sepsis is a major cause of morbidity and mortality in humans and other animals. It is estimated that 400,000-500,000 episodes of sepsis resulted in 100,000-175,000 human deaths in the U.S. alone in 1991. Sepsis has become the leading cause of death in intensive care units among patients with non-traumatic illnesses. [G. W. Machiedo et al., Surg. Gyn. & Obstet. 152:757-759 (1981).] It is also the leading cause of death in young livestock, affecting 7.5-29% of neonatal calves [D. D. Morris et al., Am. J. Vet. Res. 47:2554-2565 (1986)], and is a common medical problem in neonatal foals. [A. M. Hoffman et al., J. Vet. Int. Med. 6:89-95 (1992).] Despite the major advances of the past several decades in the treatment of serious infections, the incidence and mortality due to sepsis continues to rise. [S. M. Wolff, New Eng. J. Med. 324:486-488 (1991).]
Sepsis is a systemic reaction characterized by arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea and organ dysfunction. Sepsis can result from septicemia (i.e., organisms, their metabolic end-products or toxins in the blood stream), including bacteremia (i.e., bacteria in the blood), as well as toxemia (i.e., toxins in the blood), including endotoxemia (i.e., endotoxin in the blood). The term "bacteremia" includes occult bacteremia observed in young febrile children with no apparent foci of infection. The term "sepsis" also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitemia (i.e., helminthic or protozoan parasites in the blood). Thus, septicemia and septic shock (acute circulatory failure resulting from septicemia often associated with multiple organ failure and a high mortality rate) may be caused by a number of organisms.
The systemic invasion of microorganisms presents two distinct problems. First, the growth of the microorganisms can directly damage tissues, organs, and vascular function. Second, toxic components of the microorganisms can lead to rapid systemic inflammatory responses that can quickly damage vital organs and lead to circulatory collapse (i.e., septic shock) and oftentimes, death.
There are three major types of sepsis characterized by the type of infecting organism. Gram-negative sepsis is the most common and has a case fatality rate of about 35%. The majority of these infections are caused by Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens such as the staphylococci and streptococci are the second major cause of sepsis. The third major group includes the fungi, with fungal infections causing a relatively small percentage of sepsis cases, but with a high mortality rate.
Many of these infections are acquired in a hospital setting and can result from certain types of surgery (e.g., abdominal procedures), immune suppression due to cancer or transplantation therapy, immune deficiency diseases, and exposure through intravenous catheters. Sepsis is also commonly caused by trauma, difficult newborn deliveries, and intestinal torsion (especially in dogs and horses).
A well established mechanism in sepsis is related to the toxic components of gram-negative bacteria. There is a common cell-wall structure known as lipopolysaccharide (LPS) that is widely shared among gram-negative bacteria. The "endotoxin" produced by gram-negative organisms is comprised of three major structures: a lipoprotein; a lipid (lipid A), thought to be responsible for most of the biological properties of endotoxin; and polysaccharide structures unique to each species and distinct strains of bacteria. [D. C. Morrison, Rev. Infect. Dis. 5(Supp 4):S733-S747 (1983).] Research over the past decade or so has demonstrated that purified endotoxin can elicit all of the features of full-blown gram-negative bacteremia. Furthermore, several of the host responses to endotoxin have been identified. Two key mediators of septic shock are tumor necrosis factor (TNF) and interleukin-1 (IL-1) which are released by macrophages and appear to act synergistically in causing a cascade of physiological changes leading to circulation collapse and organ failure. [R. C. Bone, Ann. Intern. Med. 115:457-469 (1991).] Indeed, large doses of TNF [K. J. Tracey et al., Science 234:470-474 (1986)] and/or IL-1 [A. Tewari et al., Lancet 336:712-714 (1990)] can mimic the symptoms and outcome of sepsis.
It is generally thought that the distinct cell wall substances of gram-positive bacteria and fungi trigger a similar cascade of events, although the structures involved are not as well studied as gram-negative endotoxin.
Regardless of the etiologic agent, many patients with septicemia or suspected septicemia exhibit a rapid decline over a 24-48 hour period. Thus, rapid methods of diagnosis and treatment delivery are essential for effective patient care. Unfortunately, a confirmed diagnosis as to the type of infection traditionally requires microbiological analysis involving inoculation of blood cultures, incubation for 18-24 hours, plating the causative organism on solid media, another incubation period, and final identification 1-2 days later. Therefore, therapy must be initiated without any knowledge of the type and species of the pathogen, and with no means of knowing the extent of the infection.
A. Antibiotics
Antibiotics of enormously varying structure [B erdy in Advances in Applied Microbiology, (D. Perlman, ed.), Academic Press, New York, 18:309-406 (1974)] are widely used to prevent and control infections. Nonetheless, up to one half of the patients in whom bacteremia develops in the hospital die (i.e., nosocomial or iatrogenic bacteremia). [D. G. Maki, Am. J. Med. 70:719-732 (1981).] The causes for this are many-fold. First, for many commonly used antibiotics, antibiotic resistance is common among various species of bacteria. This is particularly true of the microbial flora resident in hospitals, where the organisms are under constant selective pressure to develop resistance. Furthermore, in the hospital setting, spread of antibiotic-resistant organisms is facilitated by the high density of potentially infected patients and the extent of staff-to-staff and staff-to-patient contact. Second, those antibiotics that are the most economical, safest, and easiest to administer may not have a broad enough spectrum to suppress certain infections. For example, many antibiotics with broad spectra are not deliverable orally and physicians are reluctant to place patients on intravenous lines due to the enhanced risk of infection. Third, antibiotics can be toxic to varying degrees including causing allergy, untoward interactions with other drugs, and direct damage to major organs (e.g., kidneys, liver). Many potent antibiotics are eliminated from routine use due to the probability of adverse reactions at therapeutic doses. Fourth, many antibiotics alter the normal intestinal flora and frequently cause diarrhea and nutritional malabsorption; some may even unleash opportunistic organisms which can cause life-threatening infections of the gastrointestinal (GI) tract such as Clostridium difficile. For example, antimicrobial-associated pseudomembranous colitis caused by C. difficile is a potentially serious complication associated with administration of certain antimicrobials. Physicians must therefore consider the impact of prophylactic antibiotic use on the development of resistant organisms, on overall patient health, and on the economics of health care.
While many infections are controlled by antibiotics, gram-negative bacteremia presents some special challenges. It has been shown that treatment of bacteria with antibiotics may catalyze the release of endotoxin from dying cells as their cell walls disintegrate. In experimental E. coli sepsis in rabbits, antibiotics cause a 10 to 2,000 fold increase in endotoxin levels despite decreasing levels of bacteremia. [J. L. Shenep and K. A. Morgan, J. Infect. Dis. 150:380-388 (1984).] Thus, once gram-negative bacteremia is established, there is justifiable concern that antibiotic therapy may augment symptoms while mitigating the infection.
Fortunately, certain antibiotics are known to neutralize the action of endotoxin. The polymyxin antibiotics, most notably polymyxin B and polymyxin E (also known as colistin) are cyclic polypeptide compounds produced by certain strains of Bacillus polymyxa. These antibiotics bind to the lipid A portion of endotoxin [D. C. Morrison and D. M. Jacobs, Immunochem. 13:813-818 (1976)] and neutralize its activity as measured by lethality tests in animals [D. Rifkind and J. D. Palmer, J. Bacteriol. 92:815-819 (1966)], activation of serum complement [D. C. Morrison and D. M. Jacobs, Infect. Immun. 13:298-301 (1976)], and the Limulus amebocyte lysate (LAL) assay. [M. S. Cooperstock, Antimicrob. Agents Chemother. 6:422-425 (1974).] Unfortunately, the polymyxins are not absorbed from the GI tract and must be administered parenterally. At the recommended therapeutic dose for systemic infection by P. aeruginosa (1-2.5 mg/kg body weight/day), there is a significant risk of renal impairment. [Physicians' Desk Reference, 47th Ed., pp. 818-819 (1993).] This is a major concern in patients already suffering from kidney disease. In addition to nephrotoxicity, neurotoxic reactions have been observed, the most severe being respiratory paralysis when given soon after anesthesia and/or muscle relaxants. Polymyxin B, in its intravenous form, is only given to hospitalized patients under constant supervision and monitoring of renal function. As such, polymyxins are not used routinely for systemic infections (but they are quite common as components of topical ointments).
Several approaches have been taken to reduce the toxicity of polymyxins. Colistin exhibits a lower systemic toxicity, and when complexed as methanesulfonate salt, the locally severe pain experienced at intramuscular injection sites is diminished. The toxicity of polymyxin B is also reduced by attachment to dextran, a high molecular weight carrier. [D. A. Handley, Eur. Patent Appl. Pub. No. 428486.] Conjugation to dextran is often used in an attempt to decrease the toxicity and/or increase the circulating half-lives of drugs. [P. E. Hallaway et al., Proc. Natl. Acad. Sci. USA 86:10108-10112 (1989); M. J. Poznansky and L. G. Cleland in Drug Delivery Systems: Characteristics and Biomedical Applications, (R. L. Juliano, ed.), Oxford University Press, New York, pp. 253-315 (1980); L. Molteni in Drug Carriers in Biology and Medicine, (G. Gregoriadis, ed.), Academic Press, New York, pp. 107-125 (1979); C. Larsen, Adv. Drug Delivery Rev. 3:103-154 (1989); A. D. Virnik et al., Russian Chem. Rev. 44:588-602 (1975); and Hager et al., French Patent No. 2,342,740 (1977).] Alone, polymyxin B has a half-life of only a few hours [G. Brownlee et al., Brit. J. Pharmacol. 7:170-188 (1952)], while dextran (M. W. 70,000) has a half-life in humans of about a day, depending upon the dose infused. [Reynolds et al., in Martindale - The Extra Pharmacopoeia, 28th Ed., The Pharmaceutical Press, London, pp. 512-513 (1982); and W. A. Gibby et al., Invest. Radiol. 25:164-172 (1990).]
Polymyxin B has been investigated as a specific therapy for gram-negative sepsis or endotoxemia over the past 20 years in both animal models and human trials but with mixed results. For example, endotoxin-induced disseminated intravascular coagulation (DIC) was not prevented in rabbits administered polymyxin B fifteen (15) minutes after endotoxin challenge. [J. J. Corrigan, Jr. and B. M. Bell, J. Lab. Clin. Med. 77:802-810 (1971).] In fact, most experimental studies have shown a requirement for premixture of endotoxin and polymyxin B, or administration of polymyxin B prior to endotoxin challenge to reduce or abolish the effects of endotoxin. [D. Rifkind and J. D. Palmer, J. Bact. 92:815-819 (1966); J. J. Corrigan, Jr. and B. M. Bell, J. Lab. Clin. Med. 77:802-810 (1971); B. Hughes et al., Br. J. Pharmac. 74:701-707 (1981); J. J. Corrigan, Jr. and J. F. Kiernat, Pediat. Res. 13:48-51 (1979); G. Ziv and W. D. Schultze, Am. J. Vet. Res. 44:1446-1450 (1982); and G. Baldwin et al. J. Infect. Dis. 164:542-549 (1991).] Some studies have found little benefit in polymyxin B, even as a pretreatment. [A. H. L. From et al., Infect. Immun. 23:660-664 (1979).] Importantly, clinical studies on endotoxemia in human obstructive jaundice found no benefit in polymyxin B therapy [C. J. Ingoldby et al., Am. J. Surgery 147:766-771 (1984)], consistent with results in animal models. [C. J. H. Ingoldby, Br. J. Surg. 67:565-567 (1980).]
Low dose polymyxin B therapy has also been investigated in animals and humans. In the infant rat, subinhibitory doses of polymyxin B, administered 12 hours after infection with live Haemophilus influenzae Type B organisms alone or in combination with a large dose of ampicillin, significantly reduced mortality due to the infection. The theory here is that the polymyxin B neutralizes endotoxin released by organisms killed by other antibiotics. [J. W. Walterspiel et al., Pediat. Res. 20:237-241 (1986).] It should be noted that the design of this experiment differed from the endotoxin challenge experiments, in that live organisms, not free endotoxin were the starting materials for the challenge. In humans, continuous infusion of subtherapeutic doses of polymyxin B (10-50% of normal dosage) was found to reduce endotoxin levels, restore some immune functions, and apparently (i.e., results were not statistically significant) reduce wound infection in burn patients. [A. M. Munster et al., J. Burn Care Rehab. 10:327-330 (1989).]
B. Immunization
In addition to antibiotic research and development, the effort to control bacterial infections has focused on the role of host defenses, and in particular, the humoral immune system. The role of active immunization against bacterial components and the utility of passive immunization with antibodies or plasma derived from immunized donors is a highly controversial area. While there is abundant experimental evidence that specific antibodies can protect experimental animals from infections and toxin challenge, the nature and degree of this protection and its relevance to in vivo infection is not clear despite the large volume of literature on the subject. [J. D. Baumgartner and M. P. Glauser, Rev. Infect. Dis. 9:194-205 (1987); and E. J. Ziegler, J. Infect. Dis. 158:286-290 (1988).] Disease progression in the critically ill patient, and its prevention, involves a myriad of factors which complicate the design and interpretation of human clinical trials.
In gram-negative bacteremia and endotoxemia, it was found that the frequency of septic shock was inversely related to the titer of antibodies cross-reactive with shared antigens of bacterial LPS. [W. R. McCabe et al., New Eng. J. Med. 287:261-267 (1972).] Given this correlation, an enormous effort has been expended to develop a means of raising endotoxin antibody titers and/or passively transferring endotoxin antibody from donors to experimental subjects and patients.
Antibodies to endotoxin have two important functions. First, by binding free endotoxin, antibodies may block endotoxin activity or remove it from the circulation. Second, immunoglobulin effector functions such as complement fixation and binding to Fc receptors on phagocytes can mediate killing and opsonophagocytosis of bacteria. Thus, endotoxemia, bacteremia, and the onset of sepsis, may be thwarted by such antibodies.
i) Active Immunization
One approach to protecting animals and humans from endotoxin-mediated effects is by immunization with bacteria or LPS. For example, it has been shown that immunization of rabbits with a mutant E. coli strain (J5) which lacks certain polysaccharide side chains but possesses a widely shared core lipid A structure can protect the animals from challenge with live Pseudomonas. [A. I. Braude et al., J. Infect. Dis. 136(Supp):S167-S173 (1977).] The J5 vaccine was found to be only weakly protective in a guinea pig model of Pseudomonas pneumonia, whereas a species-specific Pseudomonas LPS was greatly protective. [J. E. Pennington and E. Menkes, J. Infect. Dis. 144:599-603 (1981).] These results suggest that species-specific vaccines may be superior to cross-protective antigens for immunization of humans and other animals against endotoxin. Unfortunately, the vast diversity of LPS antigens makes the former an unlikely prospect.
While active immunization against endotoxin continues to be investigated, there are some important limitations to this approach. First, endotoxin is weakly immunogenic, eliciting only a three- to five-fold increase in antibody titers to LPS with virtually no booster response. [E. J. Ziegler et al., New. Eng. J. Med. 307:1225-1230 (1982).] Second, many patients at risk for sepsis are immunocompromised and may not be capable of mounting and/or sustaining a sufficient response to be protective upon administration of vaccine. And third, the degree of cross-protection afforded by immunization with one or more core glycolipid antigens is not well understood clinically.
ii) Passive Immunization
In order to overcome some of the limitations inherent to active immunization, various techniques have been used to produce endotoxin-binding antibodies that could be passively transferred to experimental animals or human subjects. A large number of endotoxin antibodies have been prepared by: (i) immunization of animals or humans with bacteria, LPS, or derivatives thereof and collection of immune serum or plasma; or (ii) production of monoclonal murine or human antibodies and collection and purification of these antibodies by established methods.
The two major antibody types elicited by either procedure are IgM and IgG antibodies. These antibodies differ in important aspects of their structure and effector functions as well as their titer in normal and hyperimmune plasma. Most studies suggest that IgM antibodies, by virtue of their greater avidity are more effective than IgG antibodies at protecting animals [W. R. McCabe et al., J. Infect. Dis. 158:291-300 (1988)] and humans [Id.; E. J. Ziegler et al., New. Eng. J. Med. 307:1225-1230 (1982)] from gram-negative bacteremia or endotoxin challenge. However, it should be noted that numerous IgG preparations from immunized animal donors have been developed and demonstrated to have some protective effect in experimental studies. [D. L. Dunn et al., Surgery 96:440-446 (1984); and S. J. Spier et al., Circulatory Shock 28:235-248 (1989).] The advantage to IgG preparations is that IgG titers may increase in response to repeated immunization whereas IgM titers are relatively constant. No matter what the immunization course, however, the total amount of bacterially-reactive or endotoxin-reactive antibodies in hyperimmune plasma or serum is only a small fraction of total antibody and is highly variable from donor to donor.
In order to develop more consistent preparations of therapeutic antibodies, numerous LPS-reactive monoclonal antibodies have been developed to both shared and unique epitopes. Since gram-negative sepsis can be caused by a number of species, emphasis has been placed on widely cross-reactive antibodies as potential therapeutics. Two IgM monoclonal antibodies have received the most study. A human-derived antibody now known as Centoxin-HA-1A [N. N. H. Teng et al., Proc. Natl. Acad. Sci. USA 82:1790-1794 (1985)] and a mouse-derived antibody now known as XOMEN-E5 [Young and Alam, U.S. Pat. No. 4,918,163] have been tested in both animals and humans. The animal data suggest that both antibodies are capable of binding endotoxin, neutralizing its biological activity, and suppressing gram-negative bacteremia. Unfortunately, the human clinical studies have not yielded clear benefits [E. J. Ziegler et al., New. Eng. J. Med. 324:429-436 (1991); R. L. Greenman et al., JAMA 266:1097-1102 (1991)] despite the optimism of the authors and sponsors of these trials. The U.S. Food and Drug Administration has refused to approve either antibody for the treatment of sepsis based upon the extensive clinical trials performed to date.
It should be noted that each antibody was tested in humans after the onset of symptoms of sepsis and when the type of organism was uncertain. It is widely believed that anti-endotoxin antibody treatment administered after sepsis is established may yield little benefit because these antibodies cannot reverse the inflammatory cascade initiated by endotoxin and the attendant triggering of mediators such as TNF and IL-1. In addition, the high cost of each antibody (Centoxin HA-1A was expected to cost $3700 per 100 mg dose) would limt physicians' use of aproduct where no clear benefit has been demonstrated. [K. A. Schulman et al., JAMA 266:3466-3471 (1991).] Of course, these endotoxin antibodies only target gram-negative sepsis; no equivalent antibodies exist for the array of gram-positive organisms and fungi.
With new knowledge regarding the effects of endotoxin on host inflammatory responses, other therapies are being targeted towards blockage of IL-1 and TNF functions. For example, an IL-1 receptor antagonist has been identified that occupies the same receptor site as IL-1, but mediates no biological effect. Blockage of the IL-1 receptor with this molecule can reduce mortality from endotoxin shock. [K. Ohlsson et al., Nature 348:550-552 (1990).] While the IL-1 receptor antagonist appears to be well-tolerated, the required dosage is extremely large (over 100 mg of recombinant protein per kg of body weight is infused over a period of hours to days). For human therapy, the 8-10 grams of recombinant protein anticipated to be required is likely to be extremely costly (several thousand dollars).
TNF therapies target removal of this mediator from the circulation. Monoclonal antibodies have been found to offer some protection in experimental animals [S. M. Opal et al., J. Infect. Dis. 161:1148-1152 (1990)] but studies in human patients with sepsis have not been conclusive. Once again, these antibodies are likely to be expensive therapeutic agents administered only when signs of sepsis are present.
Since the treatment of ongoing septicemia presents so many challenges, there have been several attempts at prevention. These attempts have provided mixed results. One promising study utilized hyperimmune plasma against core glycolipid in surgical patients at high risk of infection. While antibody prophylaxis did not lower the infection rate, it did reduce the severity of gram-negative infections and improved the survival of such patients. [J.-D. Baumgartner et al., Lancet 2:59-63 (1985).] Numerous studies using intravenous immunoglobulin, collected from large numbers of normal donors and containing a wide range of antibodies, have given mixed results. [J. D. Baumgartner and M. P. Glauser, Rev. Infect. Dis. 9:194-205 (1987).] The primary limitations to these studies would appear to be the variable and relatively low potency of pooled immunoglobulin preparations that were used. [T. Calandra et al., J. Infect. Dis. 158:312-319 (1988).]
Monoclonal antibodies have also been made. While these preparations should possess greater potency, their high cost, immunogenicity [S. Harkonen et al., Antimicrob. Agents Chemother. 32:710-716 (1988)] and unusually short circulating half-lives (less than 24 hr) [S. Harkonen et al., Antimicrob. Agents Chemother. 32:710-716 (1988); and C. J. Fisher et al., Clin. Care Med. 18:1311-1315 (1990)] make them unattractive candidates for prophylaxis.
Clearly, there is a great need for agents capable of preventing and treating sepsis. These agents must be capable of neutralizing the effects of endotoxin in gram-negative sepsis as well as controlling and reducing bacteremia. It would be desirable if such agents could be administered prophylactically in a cost-effective fashion. Furthermore, approaches are needed to combat all forms of sepsis, not just gram-negative cases.